Reproduction of native tissues in vitro is important as a tool, as it both enables investigation of fundamental biological processes, and drug and toxicity screenings. In order to closely mimic complex tissues in vitro, artificial multicellular systems are created from different cell types in spatially ordered structures or welldefined geometries in a 3D microenvironment. [1-3] These systems can be built from building blocks [4-7] such as cell sheets, [8] cell-laden microgels, [5] cell spheroids, [9] and organoids. [10,11] Precise control of cellular composition and spatial distribution of building blocks within artificial multicellular systems allows for reconstitution of native tissues in their healthy and disease state in vitro. [12] There are a number of methodologies developed for fabrication of complex 3D cell systems in vitro. [3-7,13,14] Directed assembly allows manual positioning or stacking building blocks to form 3D architectures. [15,16] Birey et al. applied this method for fusion of two forebrain organoids in order to mimic the human brain development and demonstrate inter-neuronal migration. [15] The method of directed assembly is, however, manual and not compatible with high throughput. Remote assembly, such as, acoustic node, [14,17] magnetic cell levitation, [13,18] optical tweezers, [19] or laser-guided direct writing, [20] can achieve assembly of cells or spheroids against gravity or viscous forces. Chen et al. demonstrated the assembly of hepatic organoids by an acoustic node technique, and the technique was able to achieve formation of bile canaliculi networks resembling native hepatic tissue. [17] Souza et al. used magnetic cell levitation to manipulate a glioblastoma cell spheroid, and a human astrocyte spheroid in order to create a cell invasion model. [18] These methods depend on sophisticated equipment, paramagnetic media, or introduce a risk of laser-induced cell damage. Another common strategy used to fabricate multicellular architecture is assembly of cell-laden hydrogels or microgels, [5,21,22] or cell seeding on scaffolds. [7,23] However, these biomaterial-based methods failed to provide high cell packing density. The use of artificial scaffolds or gel matrices additionally lead to disadvantages for constructing 3D tissue models due to their influence on cell-cell interactions, autocrine, and paracrine signaling. 3D printing [3] is a promising method for designing and achieving multicellular architectures, but it is relatively slow, not always compatible with high throughput and relies on printable bio-inks for maintaining 3D structure and cell viability. Artificial multicellular systems are gaining importance in the field of tissue engineering and regenerative medicine. Reconstruction of complex tissue architectures in vitro is nevertheless challenging, and methods permitting controllable and high-throughput fabrication of complex multicellular architectures are needed. Here, a facile and high-throughput method is developed based on a tunable droplet-fusion technique, allowing prog...

A sensitive indirect competitive nanobody-based enzyme linked immunosorbent assay (Nb-ELISA) for ochratoxin A (OTA) with high resistance to cereal matrix interference was developed. Nanobodies against OTA (Nb15, Nb28, Nb32, Nb36) were expressed in E. coli cells and their thermal stabilities were compared with that of an OTA-specific monoclonal antibody 6H8. All nanobodies could still retain their antigen-binding activity after exposure to temperature 95 °C for 5 min or to 90 °C for 75 min. Nb28 that exhibited the highest sensitivity in ELISA was selected for further research. An indirect competitive ELISA based on Nb28 was developed for OTA, with an IC50 of 0.64 ng/mL and a linear range (IC20-IC80) of 0.27–1.47 ng/mL. Cereal samples were analyzed following a 2.5 fold dilution of sample extracts, showing the good resistance to matrix interference of the Nb-ELSIA. The recovery of spiked cereal samples (rice, oats, barley) ranged from 80% to 105% and the Nb-ELISA results of OTA content in naturally contamined samples were in good agreement with those determined by a commercial ELISA kit. The results indicated the reliablity of nanobody as a promising immunoassay reagent for detection of mycotoxins in food matrix and its potential in biosensor development.

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The Wingless/Int1 (Wnt) signaling system plays multiple, essential roles in embryonic development, tissue homeostasis, and human diseases. Although many of the underlying signaling mechanisms are becoming clearer, the binding mode, kinetics, and selectivity of 19 mammalian WNTs to their receptors of the class Frizzled (FZD1–10) remain obscure. Attempts to investigate Wnt-FZD interactions are hampered by the difficulties in working with Wnt proteins and their recalcitrance to epitope tagging. Here, we used a fluorescently tagged version of mouse Wnt-3a for studying Wnt-FZD interactions. We observed that the enhanced GFP (eGFP)-tagged Wnt-3a maintains properties akin to wild-type (WT) Wnt-3a in several biologically relevant contexts. The eGFP-tagged Wnt-3a was secreted in an evenness interrupted (EVI)/Wntless-dependent manner, activated Wnt/β-catenin signaling in 2D and 3D cell culture experiments, promoted axis duplication in Xenopus embryos, stimulated low-density lipoprotein receptor-related protein 6 (LRP6) phosphorylation in cells, and associated with exosomes. Further, we used conditioned medium containing eGFP-Wnt-3a to visualize its binding to FZD and to quantify Wnt-FZD interactions in real time in live cells, utilizing a recently established NanoBRET-based ligand binding assay. In summary, the development of a biologically active, fluorescent Wnt-3a reported here opens up the technical possibilities to unravel the intricate biology of Wnt signaling and Wnt-receptor selectivity.

Development and homeostasis of multicellular organisms is largely controlled by complex cell-cell signaling networks that rely on specific binding of secreted ligands to cell surface receptors. The Wnt signaling network, as an example, involves multiple ligands and receptors to elicit specific cellular responses. To understand the mechanisms of such a network, ligand-receptor interactions should be characterized quantitatively, ideally in live cells or tissues. Such measurements are possible using fluorescence microscopy yet challenging due to sample movement, low signal-to-background ratio and photobleaching. Here, we present a robust approach based on fluorescence correlation spectroscopy with ultra-high speed axial line scanning, yielding precise equilibrium dissociation coefficients of interactions in the Wnt signaling pathway. Using CRISPR/Cas9 editing to endogenously tag receptors with fluorescent proteins, we demonstrate that the method delivers precise results even with low, near-native amounts of receptors.

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Anti-idiotypic nanobodies (AId-Nbs) are novel antigens that can replace the conventional hapten–protein conjugates of small molecules toxin, serving the same function in the competitive immunoassay.

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